The management of chronic pain through mono-pharmacotherapy directed at G protein-coupled receptors using agonists such as morphine or clonidine sometimes results in adverse consequences, such as drug-induced tolerance or hypersensitivity, or prescription drug abuse. Opioid and adrenergic agonists produce analgesia synergistically at G protein-coupled receptors (GPCRs), which translates to significant pain relief with substantially lower doses and with significantly increased therapeutic indices. The objective of this application is to characterize the mechanisms underlying specific receptor subtype pairs with established synaptic localization. The central hypothesis of this application is that the mechanism underlying synergistic interactions between co-localized receptors differs from that underlying synergistic interactions between receptors residing in distinct locations. The proposed research will 1) determine how synergistic interactions occur between opioid receptor (OR) and alpha-2 adrenergic receptor (AR) agonists, and 2) examine the functional relationships between OR and AR with regards to nociceptive neurotransmitters. Two Specific Aims comprise this application: 1. Establish the mechanism by which presynaptically co-localized GPCR pairs synergize. 2. Determine the mechanism by which presynaptic-postsynaptic GPCR pairs synergize. The mechanisms underlying synergistic interactions between GPCRs have been minimally investigated, have significant translational potential, and are, therefore, highly innovative. We and others have established that alpha 2 adrenergic and delta opioid receptors manifest powerful analgesic synergy when co-activated; more recently, we have demonstrated that co-localized alpha 2 adrenergic and delta opioid receptor subtypes can synergize in production of analgesic synergy and manifest this via cooperative inhibition of nociceptor transmitter release. The proposed research is expected to identify cellular signaling mechanisms operating in these spinal nociceptive terminals that give rise to synergy. Further, electrophysiological study of dorsal horn neurons will examine the intercellular mechanisms of drug action and interaction at the single cell level, specifically targeting two receptors that do not co-localize. The information gained from these studies may identify new therapeutic analgesic combinations and combination receptor targets leading toward reduction of dependence liabilities of analgesic treatments.